U.S. patent application number 10/137057 was filed with the patent office on 2002-11-07 for method of treating androgen-dependent disorders.
This patent application is currently assigned to Schering Corporation. Invention is credited to Bond, Richard W., Pachter, Jonathan A., Wang, Lynn.
Application Number | 20020165195 10/137057 |
Document ID | / |
Family ID | 26834877 |
Filed Date | 2002-11-07 |
United States Patent
Application |
20020165195 |
Kind Code |
A1 |
Wang, Lynn ; et al. |
November 7, 2002 |
Method of treating androgen-dependent disorders
Abstract
Novel methods of treating subjects afflicted with an
androgen-dependent disorder, such as prostate cancer and benign
prostatic hyperplasia are disclosed. Specifically, methods of
treating androgen-dependent disorders by introducing a polypeptide
or a polynucleotide encoding the polypeptide, which enhances
inactivation of active androgens, are described.
Inventors: |
Wang, Lynn; (Fanwood,
NJ) ; Bond, Richard W.; (Union, NJ) ; Pachter,
Jonathan A.; (Maplewood, NJ) |
Correspondence
Address: |
SCHERING-PLOUGH CORPORATION
PATENT DEPARTMENT (K-6-1, 1990)
2000 GALLOPING HILL ROAD
KENILWORTH
NJ
07033-0530
US
|
Assignee: |
Schering Corporation
|
Family ID: |
26834877 |
Appl. No.: |
10/137057 |
Filed: |
May 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60287756 |
May 1, 2001 |
|
|
|
Current U.S.
Class: |
514/44R ;
424/94.4 |
Current CPC
Class: |
A61K 48/00 20130101;
A61K 38/443 20130101; A61K 2300/00 20130101; C12N 15/86 20130101;
A61K 38/443 20130101; C12N 2710/10343 20130101 |
Class at
Publication: |
514/44 ;
424/94.4 |
International
Class: |
A61K 048/00; A61K
038/44 |
Claims
We claim:
1. A method for treating an androgen-dependent disorder, comprising
administering to a patient suffering from the androgen-dependent
disorder an effective amount of a polypeptide or a polynucleotide
encoding the polypeptide which enhances inactivation of an active
androgen.
2. The method of claim 1 wherein the androgen-dependent disorder is
selected from the group consisting of prostate cancer, benign
prostatic hyperplasia, acne vulgaris, seborrhea, female hirsutism,
androgenic alopecia and polycystic ovary syndrome.
3. The method of claim 1 wherein the polypeptide reduces
5.alpha.-dihydrotestosterone to
5.alpha.-androstane-3.alpha.-17.beta.-dio- l.
4. The method of claim 3 wherein the polypeptide is selected from
the group consisting of a 3.alpha.-hydroxysteroid dehydrogenase
(3.alpha.-HSD) enzyme, a 3.alpha.-HSD type 1 enzyme, a 3.alpha.-HSD
type 2 enzyme and a 3.alpha.-HSD type 3 enzyme.
5. The method of claim 1 wherein the polypeptide reduces
5.alpha.-dihydrotestosterone to
5.alpha.-androstane-3.beta.-17.beta.-diol- .
6. The method of claim 5 wherein the polypeptide is a
3.beta.-hydroxysteroid dehydrogenase (3.beta.-HSD) enzyme.
7. The method of claim 1 wherein the polypeptide oxidizes
testosterone to androst-4ene-3,17-dione or oxidizes
5.alpha.-dihydrotestosterone to 5.alpha.-androstane-3,17-dione.
8. The method of claim 7 wherein the polypeptide is selected from
the group consisting of an oxidative 17.beta.-hydroxysteroid
dehydrogenase (17.beta.-HSD) enzyme, an oxidative 17.beta.-HSD type
2 enzyme, an oxidative 17.beta.-HSD type 4 enzyme and an oxidative
17.beta.-HSD type 6 enzyme.
9. The method of claim 1 wherein the polypeptide conjugates one or
more glucuronide moieties to an androgen.
10. The method of claim 9 wherein the polypeptide is selected from
the group consisting of an uridine diphosphoglucuronosyltransferase
(UGT) enzyme, an UGT class 2 enzyme, an UGT class 2B enzyme, an
UGT2B15 enzyme, an UGT2B17 enzyme and an UGT2B20 enzyme.
11. The method of claim 1 further comprising administering an
active agent.
12. The method of claim 1 wherein the polypeptide is administered
systemically, regionally, or locally.
13. The method of claim 1 wherein the polypeptide or the
polynucleotide is administered in a multiplicity of treatments.
14. The method of claim 1 wherein the polynucleotide is part of an
expression cassette.
15. The method of claim 14 wherein the expression cassette
comprises a prostate-specific promoter.
16. The method of claim 14 wherein the expression cassette
comprises a non-prostate-specific promoter.
17. The method of claim 16 wherein the non-prostate-specific
promoter is selected from the group consisting of a cytomegalovirus
(CMV), a simian virus 40 (SV40), and a long-terminal repeat (LTR)
promoter.
18. The method of claim 14 wherein the expression cassette is part
of a vector.
19. The method of claim 18 wherein the vector is selected from the
group consisting of a recombinant adeno-associated viral vector, a
recombinant adenoviral vector, a recombinant retroviral vector, a
recombinant simian viral vector and a recombinant lentiviral
vector.
20. The method of claim 14 wherein the expression cassette is
administered as a composition comprising viral particles, wherein
the viral particles administered per treatment are in a dose
ranging from about 10.sup.9 to about 10.sup.13.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application Serial No. 60/287,756 filed May 1, 2001.
[0002] All references cited herein are incorporated in their
entirety by reference.
TECHNICAL FIELD OF INVENTION
[0003] The present invention relates to methods of using
polypeptides or polynucleotides that encode polypeptides which
enhance inactivation of active androgens for the treatment of
androgen-dependent disorders. Specifically, the present invention
relates to methods of treating conditions wherein androgen activity
is implicated such as prostate cancer, benign prostatic hyperplasia
and other androgen-dependent disorders such as androgenic
alopecia.
BACKGROUND OF THE INVENTION
[0004] An androgen-dependent disorder refers to any disorder that
can benefit from a decrease in androgen stimulation and includes
pathological conditions that depend on androgen stimulation. An
androgen-dependent disorder can result from an excessive
accumulation of testosterone or other androgenic hormone; increased
sensitivity of androgen receptors to androgen; or an increase in
androgen-stimulated transcription. Examples of androgen-dependent
disorders include prostate cancer, benign prostatic hyperplasia,
acne vulgaris, seborrhea, female hirsutism, androgenic alopecia
(which includes female and male pattern baldness), and polycystic
ovary syndrome. Early attempts to provide a chemotherapeutic agent
to counter the undesirable results of androgen-dependent disorders
resulted in the discovery of several steroidal antiandrogens having
undesirable hormonal activities of their own. The estrogens, for
example, not only counteract the effect of the androgens but have a
feminizing effect as well. Non-steroidal antiandrogens have also
been developed, for example,
4'-nitro-3'-trifluoromethyl-isobutyranilide. See Neri et al., "A
Biological Profile of a Nonsteroidal Antiandrogen, SCH 13521
(4'-Nitro-3'-Trifluoromethylisobutyranilide)", Endocrinology,
91(2):427-437 (1972). Unfortunately, even though these products are
largely devoid of direct hormonal stimulatory effects, they compete
with all natural androgens for receptor sites. Hence these products
have a tendency to feminize a male host or the male fetus of a
female host and/or initiate feedback effects that cause
hyperstimulation of the testes with increased androgen
production.
[0005] Growth of prostate tissue is androgen-dependent in benign
prostatic hyperplasia (BPH) and early stage prostate cancer.
Commonly, treatment of prostate cancer is based on surgery and/or
radiation therapy, but these methods also have deleterious side
effects and are ineffective in a significant percentage of cases.
For example, radical prostatectomy is often accompanied by a period
of dysfunction. Likewise, radiation therapy not only invokes acute
adverse effects but at times also leads to long-term complications
that can be debilitating or even life threatening, requiring more
invasive treatments or hospitalization.
[0006] Cytotoxic chemotherapy is largely ineffective in treating
prostate cancer. Even though recently developed cytotoxic agents
(e.g., paclitaxel, docetaxel, and vinorelbine) have been shown to
decrease prostate-specific antigen (PSA) and the pain secondary to
cancer, the majority of studies using chemotherapy have failed to
improve the duration of overall survival when compared to
appropriate controls. Plus, the toxicity associated with these
agents is unsuitable for treating elderly patients.
[0007] Luteinizing hormone-releasing hormone (LHRH) receptor
agonists, and more recently antagonists, are widely used for
treatment of hormone-sensitive prostate cancer. But the testicular
atrophy and the loss of libido, muscle mass and erectile function
that results from decreased gonadotropin levels is only tolerable
for life-threatening indications. Similarly, surgical castration is
an alternative for decreasing serum androgens to treat advanced
prostate cancer, but the loss of function which results can only be
considered for life-threatening conditions.
[0008] 5.alpha.-reductase inhibitors, such as finnsteride, that
inhibit reduction of testosterone to the more active androgen
5.alpha.-dihydrotestosterone (DHT) are used for the treatment of
BPH. But 5.alpha.-reductase inhibitors are only marginally
effective in treatment of BPH and often require at least six months
of treatment before efficacy may be observed. This marginal
activity may be due to prostatic accumulation of active
testosterone to eight times the normal level (Wright et al.,
"Relative Potency of Testosterone and Dihydrotestosterone in
Preventing Atrophy and Apoptosis in the Prostate of the Castrated
Rat", J. Clin. Invest., 98(11):2558-2563 (1996)).
[0009] An alternative approach involves gene therapy, that is, the
introduction of a gene into cells for therapeutic purposes. As with
more conventional therapies, the success of gene therapy relies on
targeting cells selectively and effectively without adversely
affecting other cells. For example, gene therapy using E-cadherin,
a polypeptide involved in cell-cell and cell-matrix interactions,
was proposed as a means of limiting the metastasis of cancerous
cells. This approach, however, has been unsuccessful; apparently
because other cadherins are also involved in the metastasis of
cancerous cells. In another study, patients were treated with
autologous genetically modified tumor cells that secreted
granulocyte-macrophage colony-stimulating factor (GM-CSF) in an
effort to elicit an immune response against prostate cancer
antigens. Unfortunately, only a small fraction of these patients
responded to the treatment by producing antibodies; and of those
antibodies produced, none appeared to be prostate-specific.
[0010] The present inventors have responded to the above needs by
developing novel approaches for the treatment of androgen-dependent
disorders. In contrast to other approaches such as castration, LHRH
agonists, LHRH antagonists and 5.alpha.-reductase inhibitors which
focus on decreasing synthesis of active androgens to decrease
androgen stimulation, the present invention focuses on enhancing
inactivation of active androgens, by increasing their degradation
or elimination. Surprisingly, the present invention has resulted in
tumor regression in human tumor (e.g., LNCaP tumor) xenograft
studies in mice, whereas approaches that decreased androgen
synthesis failed to induce tumor regression in this model.
Additionally, the present invention may be practiced locally
thereby decreasing androgen stimulation in the target tissue and
thus avoiding systemic side effects.
SUMMARY OF THE INVENTION
[0011] The present invention provides a method for treating an
androgen-dependent disorder comprising administering to a patient
suffering from the androgen-dependent disorder an effective amount
of polypeptide or a polynucleotide encoding the polypeptide which
enhances inactivation of an active androgen.
[0012] In a preferred embodiment, the androgen-dependent disorder
includes but is not limited to prostate cancer, benign prostatic
hyperplasia, acne vulgaris, seborrhea, female hirsutism, androgenic
alopecia, and polycystic ovary syndrome.
[0013] In one preferred embodiment, the polypeptide reduces
5.alpha.-dihydroxytestosterone (DHT) to
5.alpha.-androstane-3.alpha.-17.b- eta.-diol (3.alpha.-diol). In a
more preferred embodiment, the polypeptide is a
3.alpha.-hydroxysteroid dehydrogenase (3.alpha.-HSD) enzyme. More
preferably, the polypeptide is a 3.alpha.-HSD type 1
(3.alpha.-HSD1), a 3.alpha.-HSD type 2 (3.alpha.-HSD2), or a
3.alpha.-HSD type 3 (3.alpha.-HSD3) enzyme.
[0014] In another preferred embodiment, the polypeptide reduces
5.alpha.-dihydroxytestosterone (DHT) to
5.alpha.-androstane-3.beta.-17.be- ta.-diol (3.beta.-diol). In a
more preferred embodiment, the polypeptide is a
3.beta.-hydroxysteroid dehydrogenase (3.beta.-HSD) enzyme.
[0015] In yet another preferred embodiment, the polypeptide
oxidizes testosterone to androst-4ene-3,17-dione or oxidizes DHT to
5.alpha.-androstane-3,17-dione (5.alpha.-dione). In a more
preferred embodiment, the polypeptide is an oxidative
17.beta.-hydroxysteroid dehydrogenase (17.beta.-HSD) enzyme. More
preferably, the polypeptide is a 17.beta.-HSD type 2
(17.beta.-HSD2), a 17.beta.-HSD type 4 (17.beta.-HSD4), or a
17.beta.-HSD type 6 (17.beta.-HSD6) enzyme.
[0016] In still another preferred embodiment, the polypeptide
conjugates one or more glucuronide moieties to an androgen. In a
more preferred embodiment, the polypeptide is an uridine
diphosphoglucoronosyltransferas- e (UGT) enzyme. More preferably,
the polypeptide is a UGT class 2 enzyme or a UGT class 2B enzyme.
Still more preferably, the polypeptide is a UGT2B15, a UGT2B17 or a
UGT2B20 enzyme.
[0017] In a preferred embodiment, the present invention further
comprises administering an active agent.
[0018] In a preferred embodiment, the polypeptide is administered
systemically, regionally or locally.
[0019] In a preferred embodiment, the polypeptide or polynucleotide
is administered in a multiplicity of treatments.
[0020] In a preferred embodiment, the polynucleotide is part of an
expression cassette.
[0021] In one preferred embodiment, the expression cassette
comprises a prostate-specific promoter.
[0022] In another preferred embodiment, the expression cassette
comprises a non-prostate-specific promoter. More preferably, the
non-prostate-specific promoter is selected from the group
consisting of a cytomegalovirus (CMV), a simian virus 40 (SV40),
and a long-terminal repeat (LTR) promoter. In a more preferred
embodiment, the expression cassette containing the
non-prostate-specific promoter is administered
intraprostatically.
[0023] In a preferred embodiment, the expression cassette is part
of a vector. More preferably, the vector is selected from the group
consisting of a recombinant adeno-associated viral vector, a
recombinant adenoviral vector, a recombinant retroviral vector, a
recombinant simian viral vector and a recombinant lentiviral
vector.
[0024] In a preferred embodiment, the expression cassette is
administered as a composition comprising viral particles, wherein
the viral particles administered per treatment are in a dose
ranging from about 10.sup.9 to about 10.sup.13.
Definitions
[0025] To aid in understanding the invention, several terms are
defined below.
[0026] The term "androgen-dependent disorder" refers to any
disorder that can benefit from a decrease in androgen stimulation
and includes pathological conditions that depend on androgen
stimulation. An "androgen-dependent disorder" can result from an
excessive accumulation of testosterone or other androgenic hormone;
increased sensitivity of androgen receptors to androgen; or an
increase in androgen-stimulated transcription. Examples of
"androgen-dependent disorders" include prostate cancer, benign
prostatic hyperplasia, acne vulgaris, seborrhea, female hirsutism,
androgenic alopecia (which includes female and male pattern
baldness), and polycystic ovary syndrome.
[0027] The phrase "enhances inactivation" of an active androgen
refers to an increase in the conversion of active androgens to
inactive androgens. Alternatively, the phrase "enhances
inactivation" of active androgen refers to an increase in the
elimination of androgen. For example, elimination of androgen
results from the conjugation of one or more glucuronide moieties to
an androgen by a UGT enzyme.
[0028] The term "active androgen" refers to DHT or any other
endogenous molecule with a high affinity for the androgen receptor
which when bound induces transcription of androgen-dependent genes.
Examples of active androgens include testosterone (Kd=0.25 nM) and
DHT (Kd=0.06 nM). In contrast, the term "inactive androgen" refers
to an endogenous molecule with a relatively lower affinity for the
androgen receptor (Kd greater than 5 nM). Examples of inactive
androgens include 3.alpha.-diol (Kd=1 .mu.M) and
androst-4-ene-3,17-dione (androstenedione) (Kd=8 nM).
[0029] The term "3.alpha.-hydroxysteroid dehydrogenase
(3.alpha.-HSD)" refers to any enzyme that reduces DHT to
androstane-3.alpha.-17.beta.-dio- l (3.alpha.-diol). Examples of
"3.alpha.-HSD" include 3.alpha.-HSD type 1 enzyme, 3.alpha.-HSD
type 2 enzyme and 3.alpha.-HSD type 3 enzyme.
[0030] The term "3.beta.-hydroxysteroid dehydrogenase
(3.beta.-HSD)" refers to any enzyme that reduces DHT to
androstane-3.beta.-17.beta.-diol (3.beta.-diol).
[0031] The term "17.beta.-hydroxysteroid dehydrogenase
(17.beta.-HSD)" refers to any enzyme that oxidizes testosterone to
androst-4-ene-3,17-dione (androstenedione) or that oxidizes DHT to
androstane-3,17-dione. Examples of "17.beta.-HSD" include
17.beta.-HSD type 2 enzyme, 17.beta.-HSD type 4 enzyme and
17.beta.-HSD type 6 enzyme.
[0032] The term "uridine diphosphoglucuronosyltransferase (UGT)"
refers to any enzyme that conjugates one or more glucuronide
moieties to an androgen (e.g., testosterone and DHT) or androgen
metabolite. Examples of UGT include UGT class 2 and class 2B
enzymes (e.g., UGT2B15, UGT2B20, and UGT2B17).
[0033] The term "active agent" refers to any compound that
decreases androgenic stimulation or that has anti-tumor activity.
Examples of active agents include a P450 CYP 17 inhibitor, a
17.beta.-HSD type 3 inhibitor, a 17.beta.-HSD type 5 inhibitor, a
LHRH agonist, a LHRH antagonist, an antiandrogen and or/other
anti-tumor agents.
[0034] The term "P450 CYP 17 inhibitor" refers to any compound that
decreases the conversion of progesterone to
17.alpha.-hydroxyprogesterone and/or 17.alpha.-hydroxyprogesterone
to androstenedione. In addition, the term "P450 CYP 17 inhibitor"
refers to any compound that decreases the conversion of
pregnenolone to 17.alpha.-hydroxypregnenolone and/or
17.alpha.-hydroxypregnenolone to dihydroepiandrosterone.
[0035] The term "antiandrogen" refers to an inhibitor of the
androgen receptor. Examples of antiandrogens include flutamide,
bicalutamide and nilutamide.
[0036] The term "polynucleotide" refers to purine- and
pyrimidine-containing polymers of any length, either
polyribonucleotides or polydeoxyribonucleotides or mixed
polyribo-polydeoxyribonucleotides. This includes single- and
double-stranded molecules (i.e., DNA-DNA or DNA-RNA, and RNA-RNA
hybrids) as well as "polypeptide polynucleotides" (PNA) formed by
conjugating bases to an amino acid backbone. This also includes
polynucleotides containing modified bases including those that
permit correct read through by a polymerase while not altering
expression of a polypeptide encoded by that polynucleotide. The
term "polynucleotide" includes both the sense and antisense strands
as either an individual single strand or in the context of a
duplex.
[0037] The phrase "polynucleotide encoding" refers to a
polynucleotide that directs the expression of a specific
polypeptide. The polynucleotides include both the DNA strand
sequence that is transcribed into RNA and the RNA sequence that is
translated into polypeptide. The polynucleotides include both the
full-length polynucleotides as well as non-full length sequences
derived from the full-length sequences. It being further understood
that the sequence includes the degenerate codons of the native
sequence or sequences which can be introduced to provide a codon
preference in a specific host cell.
[0038] A "conservative substitution", when describing a polypeptide
refers to a change in the amino acid composition of the polypeptide
that does not substantially alter the polypeptide's activity. Thus,
"conservatively modified variations" of a particular amino acid
sequence refers to amino acid substitutions of those amino acids
that are not critical for polypeptide activity. Alternatively,
"conservatively modified variations" refers to substitution of
amino acids with other amino acids having similar properties (e.g.,
acidic, basic, positively or negatively charged, polar or
non-polar, etc.) such that the substitutions of even critical amino
acids do not substantially alter activity. Conservative
substitution tables providing functionally similar amino acids are
well known in the art. For example, the following six groups each
contain amino acids that are conservative substitutions for one
another:
[0039] 1) Alanine (A), Serine (S), Threonine (T);
[0040] 2) Aspartic acid (D), Glutamic acid (E);
[0041] 3) Asparagine (N), Glutamine (O);
[0042] 4) Arginine (R), Lysine (K);
[0043] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0044] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0045] See also, Voet and Voet, Biochemistry, John Wiley and Sons
(1990) pp. 59-74; Creighton, Proteins: Structure and Molecular
Properties, W.H. Freeman and Company (1984). In addition,
individual substitutions, deletions or additions which alter, add
or delete a single amino acid or a small percentage of amino acids
in an encoded sequence are also "conservatively modified
variations".
[0046] The phrase "expression cassette" refers to nucleotide
sequences that are capable of affecting expression of a gene in
compatible hosts. Expression cassettes include at minimum a
promoter and a gene transcribed from that promoter. A transcription
termination signal can also form part of the expression cassette;
and additional factors necessary or helpful in effecting expression
can also be used.
[0047] The term "administering" when referring to administering to
a patient a polypeptide or polynucleotide encoding a polypeptide,
is used herein to refer to contacting a cell with a polypeptide or
polynucleotide encoding a polypeptide such that the polypeptide or
polynucleotide is internalized into the cell. In this context,
contacting a cell with a polynucleotide is equivalent to
transferring a gene into a cell wherein the polynucleotide is
maintained episomally or is integrated into the cell's genome.
Where the drug is lipophilic or the polynucleotide is complexed
with a lipid (e.g., a cationic lipid), simple contacting will
result in transport (active, passive and/or diffusive) into the
cell. Alternatively the drug and/or polynucleotide can by itself,
or in combination with a carrier composition be actively
transported into the cell. Thus, for example, where the
polynucleotide is present in an infective vector (e.g., an
adenovirus) the vector can mediate uptake of the polynucleotide
into the cell. The polynucleotide can be complexed to agents that
interact specifically with extracellular receptors to facilitate
delivery of the polynucleotide into the cell, examples include
ligand/polycation/DNA complexes as described in U.S. Pat. Nos.
5,166,320 (Wu et al.) and 5,635,383 (Wu et al.). Additionally,
viral delivery can be enhanced by recombinant modification of the
knob or fiber domains of the viral genome to incorporate cell
targeting moieties.
[0048] The term "recombinant" refers to DNA that has been isolated
from its native or endogenous source and modified either chemically
or enzymatically to delete naturally occurring flanking nucleotides
or provide flanking nucleotides that do not naturally occur.
Flanking nucleotides are those nucleotides which are either
upstream or downstream from the described sequence or sub-sequence
of nucleotides.
[0049] A "vector" comprises a polynucleotide that can either
transiently or stably transfect or transduce a cell such that the
polynucleotide is maintained episomally or integrated within the
host cell's genome. It will be recognized that a vector can be a
naked polynucleotide, or a polynucleotide complexed with
polypeptide or lipid. The vector optionally comprises viral or
bacterial polynucleotides and/or polypeptides, and/or membranes
(e.g., a cell membrane, a viral lipid envelope). It is recognized
that vectors often include an expression cassette wherein the
polynucleotide of interest is under the control of a promoter.
Vectors include, but are not limited to replicons (e.g., plasmids,
bacteriophages) to which fragments of DNA can be attached and
become replicated. Vectors thus include, but are not limited to RNA
and autonomous self-replicating circular DNA (plasmids). Vectors
may also be of viral origin, for example, recombinant
adeno-associated viral vector, recombinant adenoviral vector,
recombinant retroviral vector, recombinant simian viral vector and
recombinant lentiviral vector. Where a recombinant microorganism or
cell culture is described as hosting an "expression vector" this
includes both extrachromosomal circular DNA and DNA that has been
incorporated into the host chromosome(s). Where a host cell is
maintaining a vector, the vector can either be stably replicated by
the cells during mitosis as an autonomous structure, or is
incorporated within the host's genome.
[0050] The term "effective amount" is intended to mean the amount
of polypeptide, polynucleotide encoding the polypeptide, or
compound which enhances inactivation of an active androgen thereby
alleviating or diminishing the symptoms or severity of the
androgen-dependent disorder.
[0051] The term "viral particles" refers to intact virions. The
concentration of infectious adenovirus viral particles is typically
determined by spectrophotometric detection of DNA, as described,
for instance, by Huyghe et al., "Purification of a Type 5
Recombinant Adenovirus Encoding Human p53 by Column
Chromatography", Hum. Gene Ther., 6(11):1403-1416 (1995).
[0052] The abbreviation "PN" as used herein, stands for "particle
number". The particle number is the total calculated number of
infectious viral particles.
[0053] The abbreviation "C.I.U." as used herein, stands for
"cellular infectious units." The C.I.U. is calculated by measuring
viral hexon polypeptide positive cells (e.g., 293 cells) after a 48
hr infection period (Huyghe et al., "Purification of a Type 5
Recombinant Adenovirus Encoding Human p53 by Column
Chromatography", Hum. Gene Ther., 6(11):1403-1416 (1995)).
[0054] The abbreviation "m.o.i." as used herein refers to
"multiplicity of infection" and is the C.I.U. per cell.
[0055] The term "tumorigenic" or "tumorigenicity" are intended to
mean having the ability to form tumors or capable of causing tumor
formation.
[0056] The term "systemic administration" refers to administration
of a composition (e.g., polypeptide) in a manner that results in
its introduction into the circulatory system. The term "regional
administration" refers to administration of a composition into a
specific anatomical space, for example, intraperitoneal,
intrathecal, subdural, or to a specific organ, and the like. The
term "local administration" refers to administration of a
composition into a limited, or circumscribed, anatomic space, for
example, intratumoral injection into a tumor mass, subcutaneous
injections, intramuscular injections, and the like. Any one of
skill in the art would understand that local administration or
regional administration can also result in entry of the composition
into the circulatory system.
DETAILED DESCRIPTION OF THE INVENTION
[0057] The present invention provides new methods of treating
androgen-dependent disorders by administering to a patient
suffering from the androgen-dependent disorder an effective amount
of a polypeptide or a polynucleotide encoding the polypeptide which
enhances inactivation of an active androgen.
[0058] The androgen pathway comprises a series of steps by which
cholesterol is converted into androgens. Following are examples of
some of the known reactions. P450 SCC converts cholesterol to
pregnenolone. Pregnenolone, in turn, may be converted to
progesterone by 3.beta.-HSD. Alternatively, pregnenolone may be
converted to 17.alpha.-hydroxypregneno- lone by CYP 17; and
17.alpha.-hydroxypregnenolone to dihydroepiandrosterone. CYP 17
also converts progesterone to 17.alpha.-hydroxyprogesterone; and
17.alpha.-hydroxyprogesterone to androst-4-ene-3,17-d ione
(androstenedione). Reductive 17.beta.-HSD isoforms reduce
androstenedione to testosterone. Conversely, oxidative 17.beta.-HSD
isoforms oxidize testosterone to androstenedione. In addition,
androstenedione can be reduced to 5.alpha.-androstane-3,17-dion- e
by 5.alpha.-reductase; and 5.alpha.-androstane-3,17-dione can be
further reduced to androsterone by 3.alpha.-HSD. Likewise,
testosterone can be reduced to DHT by 5.alpha.-reductase; and DHT
can be further reduced to androstane-3.alpha.,17.beta.-diol by
3.alpha.-reductase. Moreover, 17.beta.-HSD converts
5.alpha.-androstane-3,17-dione to/from DHT; as well as androsterone
to/from androstane-3.alpha.,17.beta.-diol. Modulation of the
androgen pathway to produce less active androgens would be
desirable for the treatment of androgen-dependent disorders.
[0059] The impact of androgen-dependent disorders can be minimized
by administering an amount of polypeptide or a polynucleotide
encoding the polypeptide which enhances inactivation of an active
androgen by converting an active androgen to an inactive androgen
thus decreasing androgen-dependent transcription. In the case of
prostate cancer or BPH, decreasing androgen-dependent transcription
would lead to a decrease in prostatic cell hyperplasia.
[0060] In one embodiment of the invention, the polypeptide is a
wild-type or modified isozyme of reductive 3.alpha.-HSD (e.g., type
1, 2, 3) which converts DHT to androstane-3.alpha.-17.beta.-diol.
In another embodiment, the polypeptide is a wild-type or modified
isozyme of oxidative 17.beta.-HSD (e.g., type 2, 4, 6), which could
be used to convert testosterone back to androstenedione.
[0061] In another embodiment of the invention, a wild-type or
modified isozyme of UGT (e.g., class 2, class 2B (UGT 2B15,
UGT2B17, UGT2B20)) could be used to transfer glucuronide to
androgens so as to enhance inactivation and elimination of
androgens.
[0062] Administration of Therapeutic Polypeptide
[0063] Polypeptides may be administered locally, regionally, or
systemically. In a preferred embodiment, polypeptides of the
present invention are administered directly into the target tissue
(e.g., intraprostatically) and in the case of prostate cancer
directly at the tumor site (i.e., intratumorally). Alternatively,
in a preferred embodiment, the polypeptide is combined with a
pharmaceutically acceptable carrier (excipient) to form a
pharmacological composition (see discussion on "Formulations"
below). The polypeptide will be administered in a therapeutically
effective dose in an amount sufficient to cure or at least
partially arrest the disorder and/or its manifestation. Amounts
effective for this use will depend upon the severity of the
disorder and the general state of the patient's health.
[0064] Administration of Therapeutic Gene
[0065] The polynucleotide encoding a therapeutic polypeptide is
preferably delivered to the target cells by a vector. Vectors can
be of non-viral (e.g., plasmids) or viral origin (e.g., adenovirus,
adeno-associated virus, retrovirus, simian virus 40, lentivirus,
herpes virus, vaccinia virus). Non-viral vectors are preferably
complexed with agents to facilitate the entry of the DNA across the
cellular membrane. Examples of such non-viral vector complexes
include formulations with polycationic agents that facilitate
condensation of DNA and lipid-based delivery systems. An example of
a lipid-based delivery system would include liposome based delivery
of polynucleotides.
[0066] In the preferred practice of the invention, the vector is a
viral vector, particularly a modified adenoviral vector. Such viral
vectors have been modified by recombinant DNA technology to enable
the expression of the polynucleotide in the target cell.
[0067] In a preferred embodiment, the polynucleotide vector
utilizes a tissue-specific promoter to express the therapeutic gene
locally in the target tissue. For example, a preferred vector for
the treatment of benign prostatic hyperplasia would utilize a
prostate-specific promoter to target expression of a therapeutic
polypeptide to prostate cells specifically. A polynucleotide vector
utilizing tissue-specific promoters could be administered
systemically, regionally, or locally. Tissue-specific promoters
include chimeric promoters. Chimeric promoters include
polynucleotides incorporating one or more tissue-specific enhancer
elements with viral promoter sequences to achieve high-level
tissue-specific expression.
[0068] In another preferred embodiment, the polynucleotide vector
utilizes a non-tissue-specific promoter. A polynucleotide vector
utilizing a non-tissue-specific promoter to drive expression of a
therapeutic polypeptide would preferably be administered regionally
or locally. For example, in the treatment of prostate cancer, a
vector utilizing a non-prostate-specific promoter would preferably
be administered intraprostatically.
[0069] Gene Transfer
[0070] The gene used in the present invention can be introduced to
the cells either as a polypeptide or as a polynucleotide. Where the
gene is provided as a polypeptide, a gene expression product (e.g.,
polypeptide or fragment thereof possessing androgen decreasing
activity) is delivered to the target cell using standard methods
for polypeptide delivery (see discussion on "Administration of
therapeutic polypeptide" above). Alternatively, the gene can be
introduced into the cell using conventional methods of delivering
polynucleotides to cells. These methods typically involve either in
vivo or ex vivo gene therapy. Particularly preferred methods of
delivery include lipid or liposome delivery and/or the use of viral
vectors (e.g., retroviral or adenoviral vectors).
[0071] Gene Therapy
[0072] In a more preferred embodiment, the polynucleotides (e.g.,
cDNA(s) encoding the therapeutic gene) are cloned into vectors that
are competent to transfer the therapeutic gene into cells (e.g.,
human or other mammalian cells) in vitro and/or in vivo. Several
approaches for introducing polynucleotides into cells in vivo, ex
vivo and in vitro have been used. For a review of gene therapy
procedures, see, e.g., Zhang and Russell, "Vectors for Cancer Gene
Therapy", Cancer Metastasis Rev., 15(3):385-401 (1996); Anderson,
"Human Gene Therapy", Science, 256(5058):808813 (1992); Nabel and
Feigner, "Direct Gene Transfer for Immunotherapy and Immunization",
Trends Biotechnol., 11 (5):211-215 (1993); Mitani and Caskey,
"Delivering Therapeutic Genes--Matching Approach and Application",
Trends Biotechnol., 11 (5):162-166 (1993); Mulligan, "The Basic
Science of Gene Therapy", Science, 260(5110):926-932 (1993);
Dillon, "Regulating Gene Expression in Gene Therapy", Trends
Biotechnol., 11 (5):167-173 (1993); Miller, "Human Gene therapy
Comes of Age", Nature 357: 455-460 (1992); Kremer and Perricaudet,
"Adenovirus and Adeno-Associated Virus Mediated Gene Transfer", Br.
Med. Bull., 51(1) 31-44 (1995); Haddada in Current Topics in
Microbiology and Immunology, Doerfler and Bohm (eds)
Springer-Verlag, Heidelberg Germany (1995); and Yu et al.,
"Progress Towards Gene Therapy for HIV Infection", Gene Ther.,
1(1):13-26 (1994).
[0073] Vectors useful in the practice of the present invention are
typically derived from viral genomes. Suitable vectors include
recombinantly modified enveloped or non-enveloped DNA and RNA
viruses, preferably selected from baculoviridiae, parvoviridiae,
picornoviridiae, herpesveridiae, poxyiridae, adenoviridiae, or
picornnaviridiae. Chimeric vectors can also be employed which
exploit advantageous merits of each of the parent vector properties
(See e.g., Feng et al., "Stable in Vivo Gene Transduction Via A
Novel Adenoviral/Retroviral Chimeric Vector", Nat. Biotechnol.,
15(9):866-870 (1997)). Such viral genomes can be modified by
recombinant DNA techniques to include the therapeutic gene and can
be engineered to be replication deficient, conditionally
replicating or replication competent. In a preferred practice of
the invention, the vectors are replication deficient or
conditionally replicating. Preferred vectors are derived from the
adenoviral, adeno-associated viral and retroviral genomes.
[0074] Conditionally replicating viral vectors are used to achieve
selective expression in particular cell types while avoiding
untoward broad spectrum infection. Examples of conditionally
replicating vectors are described in Bischoff et al., "An
Adenovirus Mutant That Replicates Selectively in p53-Deficient
Human Tumor Cells", Science, 274(5286):373-376 (1996); Pennisi,
"Will a Twist of Viral Fate Lead to a New Cancer Treatment?",
Science, 274(5286):342-343 (1996); Russell, "Replicating Vectors
for Gene Therapy of Cancer: Risks, Limitations and Prospects", Eur.
J. Cancer, 30A(8):1165-1171 (1994). Additionally, the viral genome
can be modified to include inducible promoters which achieve
replication or expression of the transgene only under certain
conditions. Examples of inducible promoters are known in the
scientific literature (See, e.g. Yoshida and Hamada,
"Adenovirus-Mediated Inducible Gene Expression through
Tetracycline-Controllable Transactivator with Nuclear Localization
Signal", Biochem. Biophys. Res. Commun., 230(2):426-430 (1997);
lida et al., "Inducible Gene Expression by Retrovirus-Mediated
Transfer of a Modified Tetracycline-Regulated System", J. Virol.,
70(9):6054-6059 (1996); Hwang et al., "A Conditional
Self-inactivating Retrovirus Vector That Uses a
Tetracycline-Responsive Expression System", J. Virol.,
71(9):7128-7131 (1997); Lee et al., "Identification of
Tumor-Specific Paclitaxel (Taxol)-Responsive Regulatory Elements in
the Interleukin-8 Promoter", Mol. Cell. Biol., 17(9):5097-5105
(1997); and Dreher et al., "Cloning and Characterization of the
Human Selenoprotein P Promoter", J. Biol. Chem.,
272(46):29364-29371 (1997). The transgene can also be under control
of a tissuespecific promoter region allowing expression of the
transgene only in particular cell types. Examples of
prostate-specific promoters include PSA and probassin (Steiner et
al., "In Vivo Expression of Prostate-Specific Adenoviral Vectors in
a Canine Model", Cancer Gene Ther., 6(5):456-464 (1999)).
Alternatively the viral vector can be modified to target its
expression to particular tissues (Printz et al., "Fibroblast Growth
Factor 2-Retargeted Adenoviral Vectors Exhibit a Modified
Biolocalization Pattern and Display Reduced Toxicity Relative to
Native Adenoviral Vectors", Hum. Gene Ther., 11(1): 191-204
(2000)).
[0075] In a particularly preferred embodiment, the therapeutic gene
is expressed in an adenoviral vector suitable for gene therapy. The
use of adenoviral vectors in vivo, and for gene therapy, is well
described in the patent and scientific literature, e.g., see,
Hermens et al., "Transient Gene Transfer to Neurons and Glia:
Analysis of Adenoviral Vector Performance in the CNS and PNS", J.
Neurosci. Methods, 71(1): 85-98 (1997); Zeiger et al., "Adenoviral
Infection of Thyroid Cells: A Rationale for Gene Therapy for
Metastatic Thyroid Carcinoma", Surgery 120(6):921-925 (1996);
Channon et al., "Adenoviral Gene Transfer of Nitric Oxide Synthase:
High Level Expression in Human Vascular Cells", Cardiovasc Res.,
32(5):962-972 (1996); Huang et al., "Gene Therapy for
Hepatocellular Carcinoma: Long-Term Remission of Primary and
Metastatic Tumors in Mice by Interleukin-2 Gene Therapy in Vivo",
Gene Ther., 3:980-987 (1996); Zepeda and Wilson, "Neonatal Cotton
Rats Do Not Exhibit Destructive Immune Responses to Adenoviral
Vectors", Gene Ther. 3(11):973-979 (1996); Yang et al., "Immunology
of Gene Therapy with Adenoviral Vectors in Mouse Skeletal Muscle",
Hum. Mol. Genet., 5:1703-1712 (1996); Caruso et al.,
"Adenovirus-Mediated Interleukin-12 Gene Therapy for Metastatic
Colon Carcinoma", Proc. Natl. Acad. Sci. USA, 93(21):11302-11306
(1996); Rothmann et al., "Heart Muscle-Specific Gene Expression
Using Replication Defective Recombinant Adenovirus", Gene Ther.,
3(10):919-926 (1996); Haecker et al., "In Vivo Expression of
Full-Length Human Dystrophin From Adenoviral Vectors Deleted of all
Viral Genes", Hum. Gene Ther., 7(15):1907-1914 (1996).
[0076] Particularly preferred adenoviral vectors include a deletion
of some or all of the polypeptide 1.times. gene. In one embodiment,
the adenoviral vectors include deletions of the E1 and/or E1
sequences. In a most preferred embodiment, the adenoviral construct
is a vector construct (e.g., pQBI-AdCHV5).
[0077] Formulations
[0078] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there are a wide variety of pharmaceutical composition formulations
suitable for the present invention.
[0079] Formulations suitable for parenteral administration, for
example, by intravenous, intradermal, and subcutaneous routes,
include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives.
[0080] Formulations suitable for injection of pharmaceutical
compositions comprising the active ingredient (polynucleotides
encoding the therapeutic polypeptide or the therapeutic polypeptide
itself) can consist of: (a) liquid solutions, for example, an
effective amount of the active ingredient suspended in diluents,
(e.g., water, saline, or PEG 400); and (b) suitable emulsions.
Formulations of the invention as injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets. In addition to the active ingredient, tablet forms can
include one or more of the following: lactose, sucrose, mannitol,
sorbitol, calcium phosphates, corn starch, potato starch,
tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal
silicon dioxide, croscarmellose sodium, talc, magnesium stearate,
stearic acid, and other excipients, colorants, fillers, binders,
diluents, buffering agents, moistening agents, preservatives, dyes,
disintegrating agents, and pharmaceutically compatible
carriers.
[0081] Formulations of the invention using polynucleotides can be
packaged in unitdose or multi-dose sealed containers, (e.g.,
ampules and vials).
[0082] The exact composition of the formulation, the concentration
of the reagents and polynucleotide in the formulation, its pH,
buffers, and other parameters will vary depending on the mode and
site of administration (e.g., whether systemic, regional or local
administration) as well as needs related to storage, handling,
shipping, and shelf life of the particular pharmaceutical
composition. Parameters can be optimized depending on the
particular formulation needed using routine methods and any of a
number of ingredients and parameters known for injectable
formulations can be used.
[0083] The dose administered to a patient, in the context of the
present invention, should be sufficient to effect a beneficial
therapeutic response in the patient over time. The dose will be
determined by the efficacy of the particular vector employed and
the condition of the patient, as well as the body weight or surface
area of the patient to be treated. The size of the dose also will
be determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
vector, or transduced cell type in a particular patient.
[0084] In determining the effective amount of vector to be
administered in treatment, the physician evaluates circulating
plasma levels of the vector, vector toxicity, progression of the
disease, and the production of anti-vector antibodies. The typical
dose for a polynucleotide is highly dependent on route of
administration and gene delivery system. Depending on delivery
method the dosage can easily range from about 1 .mu.g to 100 mg or
more. In general, the dose equivalent of a naked polynucleotide
from a vector is from about 1 .mu.g to 100 .mu.g for a typical 70
kilogram patient, and doses of vectors which include a viral
particle are calculated to yield an equivalent amount of
therapeutic polynucleotide.
[0085] For administration, transduced cells of the present
invention can be administered at a rate determined by the LD.sub.50
of the vector, or transduced cell type, and the side-effects of the
vector or cell type at various concentrations, as applied to the
mass and overall health of the patient. Administration can be
accomplished via single or divided doses as described below.
[0086] In a preferred embodiment, prior to infusion, blood samples
are obtained and saved for analysis. Vital signs and oxygen
saturation by pulse oximetry are closely monitored. Blood samples
are preferably obtained 5 minutes and 1 hour following infusion and
saved for subsequent analysis. In ex vivo therapy, leukopheresis,
transduction and reinfusion can be repeated, e.g., every 2 to 3
months. After the first treatment, infusions can be performed on a
outpatient basis at the discretion of the clinician. If the
reinfusion is given as an outpatient, the participant is monitored
for at least 4, and preferably 8 hours following the therapy.
[0087] As described above, the adenoviral constructs can be
administered systemically (e.g., intravenously), regionally (e.g.,
intraperitoneally) or locally (e.g., intraprostaticlly). Typically
such administration is in an aqueous pharmacologically acceptable
buffer as described above. However, in other embodiments, the
adenoviral constructs or the expression cassettes are administered
in a lipid formulation, more particularly either complexed with
liposomes to for lipid/polynucleotide complexes or encapsulated in
liposomes, more preferably in immunoliposomes directed to specific
tumor markers. It will be appreciated that such lipid formulations
can also be administered topically, or systemically.
[0088] Dosage
[0089] The polypeptide or polynucleotide can be administered in a
single dose or a multiplicity of treatments.
[0090] In a preferred embodiment, the polynucleotide encoding
polypeptide is delivered by a recombinant adenoviral vector
administered in a total dose ranging from about 10.sup.9 to about
10.sup.12 adenovirus particles.
[0091] Routes of Delivery
[0092] Pharmaceutical compositions can be delivered by any means
known in the art, e.g., systemically, regionally, or locally; by
intra-arterial, intratumoral, intravenous (i.v.), parenteral,
topical or local administration, as subcutaneous, intratumoral
(e.g., transdermal application or local injection). Particularly
preferred modes of administration include intraprostatic or
intratumoral injections, especially when it is desired to have a
"regional effect," e.g., to focus on a specific organ (e.g.,
prostate).
[0093] Treatment Regimens
[0094] Pharmaceutical compositions can be administered in a variety
of unit dosage forms depending upon the method of administration.
Detailed information for preparing pharmaceutical compositions can
be found in such publications as Remington's Pharmaceutical
Science, 15th ed., Mack Publishing Company, Easton, Pa.
[0095] In therapeutic applications, compositions containing the
active ingredient are administered to a patient suffering from a
disease characterized by cancer or hyperproliferation of one or
more cell types in an amount sufficient to cure or at least
partially arrest the disease and/or its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose" and will depend upon the severity of the disease as
well as the general state of the patient's health.
[0096] Single or multiple administrations of the compositions can
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the active ingredient of the
present invention to effectively treat the patient. The treatment
preferably results in a decrease in the manifestation of the
androgen-dependent disorder.
[0097] Modifications of Therapeutic Polypeptide or
Polynucleotides
[0098] One of skill in the art will appreciate that many
conservative variations of the polypeptide or polynucleotides
described herein yield functionally identical products. For
example, due to the degeneracy of the genetic code, "silent
substitutions" (i.e., substitutions of a polynucleotide which do
not result in an alteration in an encoded polypeptide) are an
implied feature of every polynucleotide which encodes an amino
acid. Similarly, "conservative amino acid substitutions," in one or
a few amino acids in an amino acid sequence are substituted with
different amino acids with highly similar properties (see, the
definitions section, supra), are also readily identified as being
highly similar to a disclosed amino acid sequence, or to a
disclosed polynucleotide which encodes an amino acid. Such
conservatively substituted variations of each explicitly described
sequence are a feature of the present invention.
[0099] One of skill would recognize that modifications can be made
to the polypeptide without diminishing its biological activity.
Some modifications can be made to facilitate the cloning,
expression, or incorporation of the targeting molecule into a
fusion polypeptide. Such modifications are well known to those of
skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site, or additional
amino acids (e.g., poly His) placed on either terminus to create
conveniently located restriction sites or termination codons or
purification sequences.
[0100] Modifications to polynucleotides and polypeptides can be
evaluated by routine screening techniques in suitable assays for
the desired characteristic. For instance, changes in the
immunological character of a polypeptide can be detected by an
appropriate immunological assay. Modifications of other properties,
for example, polynucleotide hybridization to a target
polynucleotide, redox or thermal stability of a polypeptide,
hydrophobicity, susceptibility to proteolysis, or the tendency to
aggregate are all assayed according to standard techniques.
[0101] In vitro and in vivo studies were performed on LNCaP cells,
a cell line established from a metastatic lesion of human prostatic
adenocarcinoma that is androgen responsive (Horoszewicz et al.,
Prog. Clin. Biol. Res., 37:115-132 (1980)).
[0102] Example 1 describes in vitro studies regarding the
conversion of androgens by 3.alpha.-HSD types 1 and 3 as well as
17.beta.-HSD type 2. In addition, the rate of DHT conversion by
3.alpha.-HSD isozymes was explored as well as the impact of
3.alpha.-HSD on the production of PSA.
EXAMPLE 1
[0103] In vitro Studies of Androgen Conversion by 3.alpha.-HSD and
17.beta.-HSD
[0104] LNCaP cells were transduced with recombinant adenovirus
(rAdv) rAdv-3.alpha.-HSD1 (rAdvHSD1) or rAdv-3.alpha.-HSD3
(rAdv-HSD3) and then tested for their ability to convert
.sup.14C-labeled DHT to 3.alpha.-diol or .sup.3H-labeled
3.alpha.-diol to DHT indicating predominantly reductive activities
of these 3.alpha.-HSD isoforms. Both enzymes 3.alpha.-HSD1 and
3.alpha.-HSD3 catalyzed the reductive conversion of DHT to
3.alpha.-diol. In contrast, there was no detectable conversion of
3.alpha.-diol to DHT. Although there can be differences in
expression levels, it appears that 3.alpha.-HSD1 was much more
active than 3.alpha.-HSD3 in conversion of DHT to 3.alpha.-diol.
Therefore 3.alpha.-HSD1 was chosen for proof-of-principle
studies.
[0105] Androgen conversion by 17.beta.-HSD type 2 was also examined
in vitro. In brief, high-expressing 17.beta.-HSD type 2 cell lines
were established from which cellular extracts were isolated and
incubated with radioactively labeled androgens to determine whether
androgen conversion occurred. More specifically, human embryonic
kidney (HEK) 293 cells were transfected with a DNA construct
containing human 17.beta.-HSD2 downstream of a CMV promoter and a
neomycin resistance gene downstream of an SV40 promoter
(pcDNA3-lnvitrogen). Cell lines permanently expressing human
17.beta.-HSD2 were selected with 650 .mu.g/ml G418 and verified by
both PCR as well as 17.beta.-HSD2 activity assays. A
high-expressing cell line of 17.beta.-HSD2 was selected to examine
androgen conversion. The high-expressing cell line was grown to
about 80% confluence prior to harvesting cells by trypsinization
and subsequent centrifugation. The cell pellet was then resuspended
in 20 mM phosphate buffer with protease inhibitors, 1 mM EDTA, 0.25
M sucrose, and 20% glycerol. The isolated cells were lysed by
sonication and centrifuged at 10,000.times. g to remove cellular
debris. The soluble fraction was then centrifuged at 100,000.times.
g after which the insoluble fraction was resuspended in the same
buffer as above and the polypeptide content measured (Bio-Rad
polypeptide assay kit). Various amounts of polypeptide were
incubated with 100 nM of either .sup.14C-androstenedione (A) under
reducing conditions (NADH and NADPH) or .sup.14C-testosterone (T)
under oxidizing conditions (NAD.sup.+ and NADP.sup.+) for 30
minutes at 37.degree. C. Following incubation, the resulting
products were separated by thin layer chromatography (TLC) with
chloroform:ethyl acetate (3:1) and the .sup.3H-containing compounds
quantitated using a phosphorimager. The data demonstrated that
human 17.beta.-HSD2 is much more active in oxidizing T to A (about
90% conversion at 1 mg) than in reducing A to T (barely detectable
activity at 1 mg). Therefore, 17.beta.-HSD2 is also a viable
candidate for treating androgen-dependent disorders.
[0106] For proof-of-principle studies, 3.alpha.HSD1 was chosen. The
rate of conversion of DHT to 3.alpha.-diol by 3.alpha.HSD1 was
found to be dose-dependent. The rate of conversion of DHT to
3.alpha.-diol was determined as follows: 5.times.10.sup.5 LNCaP
cells were plated per well of a 6-well tissue culture plate; 48 hrs
after cells were plated, rAdvHSD1 or control-Adv was added to
cells; labeled DHT was added at various specified intervals of
time; cells were subsequently incubated for 1 hr at 37.degree. C.
after which supernatant was collected from the individual culture
dishes and collected supernatants were extracted using a mixture of
chloroform:methanol followed by thin layer chromatography (TLC) to
separate the labeled steroids. Based on measurements from TLC
plates, the percent conversion of DHT to 3.alpha.-diol was
calculated.
[0107] LNCaP cells transduced with rAdvHSD1 were assayed for
production of PSA, a marker of prostate cancer and BPH
aggressiveness that is dependent on androgen stimulation in LNCaP
cells. PSA production was found to decrease in a dose-dependent
manner after transduction with rAdvHSD1. In contrast, transduction
with control virus had no effect on PSA production.
[0108] The effect of rAdvHSD1 on PSA production was further
examined using R1881, a non-hydrolyzable androgen, to stimulate PSA
production. In sum, rAdvHSD1 had no effect on PSA production
induced by R1881 thereby supporting the hypothesis that the effect
of rAdvHSD1 is mediated by enhanced conversion of DHT.
[0109] Next, in vivo studies were undertaken to examine whether the
results observed in vitro were indicative of decreased
androgen-dependence in vivo.
[0110] Example 2 describes in vivo studies regarding the effect on
tumor growth by 3.alpha.-HSD1. Specifically, LNCaP cells transduced
with rAdvHSD1 at different m.o.i. were implanted into SCID mice. In
another study, established LNCaP tumors were intratumorally
injected with rAdvHSD1 and harvested 21 days later to assay
3.alpha.-HSD activity.
EXAMPLE 2
[0111] In vivo Studies of Tumor Growth Using LNCaP Cells Transduced
With rAdvHSD1
[0112] To test the ability of rAdvHSD1 to inhibit the growth of
LNCaP tumors in SCID mice, LNCaP cells were transduced with
rAdvHSD1, control-Adv, or mock transduced with PBS in vitro. After
24 hrs, the cells were washed and subcutaneously injected along
with matrigel into SCID mice. LNCaP cells transduced with rAdvHSD1
and injected into SCID mice demonstrated decreased tumorigenicity
in vivo. In fact, rAdvHSD1 was able to completely inhibit tumor
growth in a dose-dependent manner while control-Adv had little or
no effect on tumor growth. The ability of rAdvHSD1 to block growth
of LNCaP cells in vivo 60 days after transduction was surprising as
rAdv expression of transgenes is usually transient in vivo.
[0113] Lastly, to test the ability of rAdvHSD1 to inhibit the
growth of established LNCaP tumors, LNCaP tumors were injected
intratumorally with rAdvHSD1, control-Adv, or PBS. Injections
started 34 days after implanting LNCaP cells subcutaneously and
were delivered three times with 48 hour intervals between each
injection. Intratumoral injection of rAdvHSD1 resulted in
regression of LNCaP tumors in a dose-dependent manner whereas
control virus had little or no effect. This dramatic tumor
regression was extremely surprising, since even the dramatic
decrease in serum androgens that follows surgical castration has
been unable to induce regression of LNCaP tumors (Gleave et al.,
"Serum Prostate Specific Antigen Levels in Mice Bearing Human
Prostate LNCaP Tumors Are Determined by Tumor Volume and Endocrine
and Growth Factors" Cancer Res, 52(6):1598-1605 (1992)).
[0114] To test for active transgene expression in tumors that had
been intratumorally injected, tumors were harvested 21 days after
the last injection with rAdvHSD1 or control-Adv and examined for
3.alpha.-HSD activity. Even after 21 days post-transduction in vivo
the harvested rAdvHSD1 treated tumor cells still had high levels of
3.alpha.-HSD activity.
[0115] The above-described examples show that gene therapy which
enhances inactivation of an active androgen is effective to treat
androgen-dependent disorders. In the model of prostate cancer
examined, 3.alpha.-HSD1 was able to decrease the level of active
androgen available thereby decreasing androgen-dependent
transcription and androgen-dependent tumor growth.
[0116] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
* * * * *